Human embryonic stem (hES) cells typically require a substrate and culture medium to maintain indefinite self-renewal and pluripotency characteristics. The most common substrates for culturing hES cells are monolayers of inactivated fibroblast feeder cells grown on tissue culture (TC) polystyrene surface or TC culturing vessels coated with an extracellular matrix (ECM), for example BD Matrigel™-coated TC culturing vessels. Both of these substrates are poorly defined and introduce a high degree of experimental variability. Since hES cells are thought to have a significant potential implication in furthering knowledge of developmental biology, drug discovery and may play an important role in future clinical applications, it is important to identify conditions for culturing these cells on defined surfaces.